Promiscuous Pseudomonas: Uptake of non-endogenous ligands for iron acquisition

Article abstract of DOI:10.1016/j.tetlet.2021.153204

Iron is an essential nutrient to nearly all living beings. However, its acquisition poses a significant challenge to many organisms, including most bacteria. One of the main iron uptake strategies employed by bacteria is the uptake of siderophores, small

Full text of DOI:10.1016/j.tetlet.2021.153204

Tetrahedron Letters xxx (xxxx) xxx
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Tetrahedron Letters
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Promiscuous Pseudomonas: Uptake of non-endogenous ligands for iron
acquisition
⇑
Anna R. Kaplan, William M. Wuest
Department of Chemistry, Emory University, Atlanta, GA 30322, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Iron is an essential nutrient to nearly all living beings. However, its acquisition poses a significant chal-
lenge to many organisms, including most bacteria. One of the main iron uptake strategies employed by
bacteria is the uptake of siderophores, small molecules that chelate extracellular iron. The pathogenic
species Pseudomonas aeruginosa produces two different siderophores, pyochelin and pyoverdine. P. aerug-
inosa senses the amount of bioavailable extracellular iron in order to regulate the production levels of
each of these two siderophores. In previous work, we found that a series of pyochelin biosynthetic shunt
products enhanced the growth of P. aeruginosa in iron- depleted conditions when prechelated with iron.
Thus, on the basis of these results, we investigated the physiochemical and biological properties of a ser-
ies of non-native oxygen counterparts to these metabolites in the current study.
Received 17 April 2021
Revised 17 May 2021
Accepted 21 May 2021
Available online xxxx
Dedicated to Prof. Dale Boger for his
contributions to the field of medicinal
chemistry and continued support of the
next generation of scientists.
Ó 2021 Elsevier Ltd. All rights reserved.
Keywords:
Iron acquisition
Siderophores
Oxazolines
Pseudomonas aeruginosa
Introduction
produce multiple siderophores, one of which is the pathogenic
bacteria Pseudomonas aeruginosa, which is known to colonize the
Iron is an essential nutrient playing a central role in numerous
vital biological processes including photosynthesis, respiration,
nitrogen fixation, oxygen transport, and DNA synthesis [1–3]. It
is the fourth most abundant element on earth, however its acqui-
sition still presents a unique challenge for many organisms includ-
ing most bacteria [1–3]. For example, in densely populated
terrestrial environments, such as the rhizosphere, most of the iron
is present as a highly insoluble ferric oxide complex (Fe2O3), limit-
ing the concentration of bioavailable iron to 10-9 to 10-18 M, which
is far below the 10-6 M required for survival [2–4]. Another partic-
ularly sparse environment is that of mammalian host organisms,
because the majority of FeIII is tightly complexed and stored in var-
ious circulating proteins such as ferretin, heme, and transferrin
thereby limiting the available FeIII concentration to a lowly 10-24
M [2,3,5].
lungs of immunocompromised patients [7]. The production levels
of its two native siderophores, high-affinity pyoverdine and lower
affinity pyochelin (structure shown in Fig. 1a) is regulated by the
ferric uptake regulatory system (Fur) based on the amount of
bioavailable extracellular iron (Fig 1a) [8,9]. In previous work, we
investigated a series of pyochelin biosynthetic shunt products
and found that when complexed with iron, these metabolites pro-
moted growth of P. aeruginosa wild-type (PAO1) and a double
mutant
(DpvdDDpchEF) incapable of producing siderophores
(Fig. 1a) [10]. Our lab has had a long standing interest in sidero-
phores [11], particularly those with scaffolds similar to pyochelin
[12]. In particular, the thiazoline/oxazoline motif akin to many
siderophores [13,14], has garnered much attention [15]. Thus, we
wanted to explore if exchanging the sulfur in these shunt metabo-
lites with an oxygen (Fig. 1b) would abolish the growth enhance-
ment effect observed previously, or if P. aeruginosa would in fact
be able to import these complexes as xenosiderophores [9]. Herein,
we synthesized a series of oxygen analogs of previously studied
metabolites and investigated their physiochemical and biological
properties.
To circumvent this issue of iron limitation, bacteria have
evolved several iron uptake strategies, the most common of which
is the uptake of siderophores: small molecules excreted by
bacteria to chelate extracellular iron and then reimported as
siderophore-iron complexes [1–4,6]. Many species of bacteria
The synthesis of this series of compounds was achieved in a
straightforward and highly efficient manner (Scheme 1). 2-
⇑
Corresponding author.
https://doi.org/10.1016/j.tetlet.2021.153204
0040-4039/Ó 2021 Elsevier Ltd. All rights reserved.
Please cite this article as: A.R. Kaplan and W.M. Wuest, Promiscuous Pseudomonas: Uptake of non-endogenous ligands for iron acquisition, Tetrahedron
Letters, https://doi.org/10.1016/j.tetlet.2021.153204

A.R. Kaplan and W.M. Wuest
Tetrahedron Letters xxx (xxxx) xxx
each corresponding compound to a solution of FeCl3, (both in
methanol) a distinct color change was observed, changing from
yellow to purple. Consistent with previous findings [10], the degree
of color change was concentration dependent, as the addition of
increasing amounts of compound to FeCl3 yielded solutions with
a darker shade of purple (Fig. S1). It should be noted that this effect
was more obvious for some compounds and less obvious for
others.
Quantification of the iron-binding properties of these com-
pounds was achieved using fluorescence titration experiments. FeIII
was added in 0.1 M equivalent increments to a solution of each
compound in methanol until a full molar equivalent had been
added. The emission spectra were then recorded after each addi-
tion, allowing us to generate fluorescence titration curves (Fig. 2a).
Similar to previous work [10], these spectra showed that the emis-
sion signals of all compounds were quenched with different
amounts of FeIII, indicating that the stoichiometry with which they
each bind iron is different. These spectra were then extrapolated to
determine ligand-to-FeIII binding ratios (L:FeIII), as well as putative
dissociation constant (Kd) values (Fig. 2b, S2). It should be men-
tioned that the Kd values were calculated in prism and serve as a
ballpark estimate of the true Kd value of each compound. Notwith-
standing, these Kd values are within 1–2 orders of magnitude and
are thus relatively comparable to those of pyochelin [19–21].
Finally, a preliminary biological investigation of this series was
performed. In our previous work, we tested our series of metabo-
lites against a panel of Pseudomonads (P. aeruginosa, P. fluorescens,
P. putida, and P. syringae) as well as other clinically relevant species
including (methicillin-resistant) Staphylococcus aureus, Enterococ-
cus faecalis, and Escherichia coli, with the hypothesis that they
may be acting through a mechanism similar to that of yersini-
abactin, wherein chelation of iron would inhibit the growth of a
given bacteria [22]. However, no such inhibition was observed
for any of the compounds against any bacterial species. Because
these oxygen-containing counterparts, unlike those studied in
our previous work, are not endogenous to P. aeruginosa, we
hypothesized that they could garner this inhibitory effect. Albeit,
no growth effect was observed, thus we moved on to elucidate
potential growth-enhancing effects of these molecules on P.
aeruginosa.
Fig. 1. (A) Summary of results from previous work; (B) Structures of compounds
studied in this work.
All four compounds were incubated (as free ligands) with both
wild type PAO1 and the previously mentioned double knockout
mutant
DpvdDDpchEF in iron depleted media and growth to sta-
tionary phase was monitored up to 20 h (Fig. 3). Unsurprisingly,
there was no noticeable influence on growth, consistent with pre-
vious findings. However, again similar to our previous work, when
these compounds were prechelated with FeIII, a marked increase in
growth was observed [10]. This enhancement of growth was sim-
ilar to that resulting from incubation with the same concentration
of free FeIII (125
pvdD pchEF, thereby indicating that this observed enhancement
lM) in the case of PAO1 and greater for
D
D
Scheme 1. Reagents and conditions: (i) AcCl, MeOH, 25 °C, 16 h, 54%; (ii) L-serine
methyl ester hydrochloride, Et3N, C2H4Cl2, 90 °C, 16 h, 45%; (iii) LiOH, THF/H2O
(1:1), 25 °C, 2 h, 50% for 1, 84% for 3; (iv) LAH, Et2O, 0 °C to 25 °C, 1 h, 54% for 2, 35%
for 4; (v) DBU, BrCCl3, CH2Cl2, 0 °C to 25 °C, 74%.
Hydroxybenzonitrile was converted to imidate S1, followed by
condensation with L-serine methyl ester hydrochloride to give oxa-
zoline S2 [16]. This material was then hydrolyzed or reduced to
give carboxylic acid 1 and primary alcohol 2, respectively. Desatu-
ration of the oxazoline was achieved utilizing methodology devel-
oped by Wipf and Williams, furnishing oxazole S3 [17,18]. In the
same fashion as with the oxazolines, hydrolysis and reduction
afforded 3 and 4, respectively.
Fig. 2. (A) Fluorescence titration curves of 1–4 titrated with 1.0 equivalents of FeCl3
(each titration was performed in triplicate); (B) Table of binding stoichiometries
and calculated Kd values for 1–4 and pyochelin [19–21].
The iron-binding properties of this series was then investigated
qualitatively and quantitatively. Upon addition of a solution of
2

A.R. Kaplan and W.M. Wuest
Tetrahedron Letters xxx (xxxx) xxx
these compounds. However, one claim that can be made is that
P. aeruginosa is fairly promiscuous in its uptake of iron-chelated
small molecules in iron-depleted laboratory conditions. Future
work is focused on determining if this phenomenon is specific to
Pseudomonas or if it applies to other pathogenic bacteria such as
Acinetobacter baumanni.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgments
We are grateful to the NIH (GM119426) and NSF (CHE2003692)
for funding this work. The NMR instrumentation used in this work
was funded by the NSF (CHE1531620). A.R.K. acknowledges the
National
Science
Foundation
Pre-Doctoral
Fellowship
(DGE1937971). The content is solely the responsibility of the
authors and does not necessarily represent the official views of
the National Institutes of Health. We also thank Prof. Steve Diggle
(Georgia Tech) for the P. aeruginosa
DpvdDDpchEF strain used in
the study and Prof. Steve Diggle and Prof. Marvin Whiteley (Geor-
gia Tech) for fruitful discussions.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tetlet.2021.153204.
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